Unveiling the hidden players: exploring the role of gut mycobiome in cancer development and treatment dynamics

ABSTRACT The role of gut fungal species in tumor-related processes remains largely unexplored, with most studies still focusing on fungal infections. This review examines the accumulating evidence suggesting the involvement of commensal and pathogenic fungi in cancer biological process, including oncogenesis, progression, and treatment response. Mechanisms explored include fungal influence on host immunity, secretion of bioactive toxins/metabolites, interaction with bacterial commensals, and migration to other tissues in certain types of cancers. Attempts to utilize fungal molecular signatures for cancer diagnosis and fungal-derived products for treatment are discussed. A few studies highlight fungi’s impact on the responsiveness and sensitivity to chemotherapy, radiotherapy, immunotherapy, and fecal microbiota transplant. Given the limited understanding and techniques in fungal research, the studies on gut fungi are still facing great challenges, despite having great potentials.


Introduction
While the gut microbiome comprises bacteria, fungi, and viruses, research on diseases has predominantly centered on understanding the interaction between the host and gut bacteria.This emphasis arises from the fact that the intestinal bacteriome constitutes more than 99.9% of the overall gut microbiome. 1 Fungi, constituting the remaining 0.1%, however, also play a crucial role in normal and pathological mechanisms, akin to bacteria.Fungi establish early colonization in the intestine during birth, breastfeeding, or through ingestion via food, respiratory tracts, or skin-mouth contact.Food is another significant fungal source.The gut mycobiota is mainly composed of Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota.Prominent commensal fungi species in the human gut include Candida spp., Saccharomyces spp., and Malassezia spp. 2 Normally nonpathogenic, these fungi can become serious threats when the host's homeostasis is disrupted, particularly in cases of a suppressed immune system or reduced bacterial population.
Fungi exhibit morphological adaptability, capable of transitioning between unicellular and multicellular forms, particularly yeast and hyphae, respectively, which influences their pathogenicity.4][5] Fungi also demonstrate the ability to generate both harmful and beneficial compounds.Utilizing toxic metabolites and proteases, fungi can switch between various morphological states, aiding in adhesion, host barrier penetration, and subversion of the host immune response.Recent studies affirm that specific gut bacteria possess the ability to trigger tumorigenesis in genetically susceptible murine models.
Certain types of tumor, especially colorectal cancer (CRC), have been extensively studied, with emphasis given to Enterotoxigenic Bacteroides Fragilis, Escherichia coli, and Fusobacterium nucleatum as key components in their pathogenesis. 6imited research has focused on the tumorigenesis of intestinal fungi.Nevertheless, existing studies suggest a potential connection between diverse cancer types and various fungal species.

Common pathogenic mechanisms
Over the decades, studies have been majorly focused on the infections and septicemia caused by gut fungi.However, there are still studies implicating the possible common mechanisms by which gut fungi are able to induce host immune and microbiome dysregulation, and subsequently leading to oncogenesis.These may include the fungihost recognition and immune regulation, biofilm formation, bacterial-fungal interactions, and toxins and metabolites produced by them (Figure 1).

Fungi-host recognition and inflammatory signaling pathway
C-type lectin receptors (CLRs), typically Dectin-1, Dectin-2, Dectin-3, and Mincle, are subtypes of pattern recognition receptors (PRRs), primarily expressed on myeloid cell surfaces including surfaces of macrophages, neutrophils, and dendritic cells.These CLRs detect fungal cell wall components like β-glucans and α-mannans, engage in sensing fungal infections and activating downstream signaling pathways, triggering inflammation and immune responses. 7This ligand-receptor binding activates multiple intracellular signaling pathways, including adaptor molecules, kinases, and transcription factors.The Caspase Recruitment Domain-containing protein9 (CARD9) is an essential adaptor protein restrictedly expressed in myeloid cells and is critical in initiating inflammatory caspase in response to Candida, Malassezia, and Aspergillus.3][14] The NLRP3 inflammasome is potentiated by diverse activators and recognizing patterns, including damage-associated molecular patterns (DAMP), and pathogen-associated molecular patterns (PAMP), which are targets of fungi, bacteria, and viruses. 15Subsequently, downstream immune response is triggered by these activators in the different circumstances, including fungal invasions and infections.
NLRP3 inflammasome activation is found to be associated with two stepwise signals.Signal 1 initiates the immune recognition response such as recognition by Dectin-1 mentioned above, followed by activation of nuclear factor-κB (NF-κB), driving the production of pro-IL-1β.The signal 2 involves subsequent activation of caspase-1, resulting in the maturation of IL-1β. 16,17ectin-3, also known as CLED4D or CLECSF8, operates as a PRR in various myeloid cells.It recognizes multiple fungi, including C. albicans and Cryptococcus, by detecting α-mannans on their surfaces.A prior study conducted by Zhu et al. revealed that Dectin-3 forms a heterodimeric complex with Dectin-2 to detect α-mannans. 180][21][22] These responses contribute to both innate and adaptive immunity. 23The inflammatory activation pathways and interferons further promote the regulation and alteration of the host immune response, playing a crucial role in the development of both malignant and benign conditions.Malassezia can elicit cooperative recognition by Mincle and Dectin-2 through distinct ligands that cause skin inflammation. 24he CLRs, accompanied by the subsequent downstream activation of variable signaling pathways, actively participate in the regulation of immune responses, and thus affecting disease progression.

Immune cell activation
Myeloid-derived suppressor cells (MDSCs), composed of granulocytic MDSCs (G-MDSCs) and monocytic MDSCs (GM-MDSCs), constituting a heterogeneous group of immature myeloid cells.Their role involves promoting immune suppression and aiding in tumor development.Fungal dysbiosis leads to an increase in the release of MDSCs, thus contributing to tumorigenesis.In CRC patients, a positive correlation occurs between the abundance of MDSCs at tumor sites and the fungal burden.][27][28][29] Th17 cells, an antigen-specific subset of CD4 + T cells, contribute to coordinating immune responses against extracellular bacteria and fungi.Th17 cells, initially activated by C. albicans, broaden their response to target other fungi through cross-reactivity.Intestinal inflammation expands C. albicans-specific and cross-reactive Th17 cells. 30Dectin-SyK-CARD9 signaling pathways can stimulate the secretion of interferons and cytokines, as well as the differentiation of T lymphocytes into IL-17-producing CD4 + T cells. 31,32Pro-inflammatory cytokine IL-17 secreted by Th17 cells is regarded as a contributor to multiple autoimmune diseases including autoimmune thyroid disease, 33 atopic dermatitis, 34 IBD, 35 etc.Studies have explored its role in tumor development in mice, revealing that indole's carcinogenic effect involves interfering the Th17 cell differentiation and IL-17 production under AhR regulation. 36Additionally, IL-23, a cytokine crucial for expanding and sustaining Th17 cells, is vital for inducing T-cell mediated colitis in murine models. 37][39] MDSCs and Th17 cells significantly promote immune suppression and tumor development.Fungal dysbiosis induces increased MDSC release and Th17 differentiation, contributing to tumor progression.Th17 cells, initially activated by C. albicans, amplified their response to target fungi and contributes to inflammation.The stimulation of stimulate Th17 differentiation and IL-17 production is assisted by the Dectin-SyK-CARD9 pathways, as implicated in autoimmune diseases and tumor development.

Bacterial-fungal interactions
Bacteria and fungi can interact synergistically, mutually, or antagonistically, shaping the microbiome's unique features in different human body parts and health states.Bacterial species are often found accompanied with fungi in the formation of polymicrobial biofilms, such as Streptococcus mutans, P. aeruginosa, and S. aureus . 40Infections originating from biofilms that involve both bacteria and fungi pose a significantly greater challenge for treatment compared to those involving a single species.Addressing these infections requires intricate multi-drug treatment approaches. 41The hostbacterial-fungal interactions can affect the survival, morphogenesis, and invasiveness of fungi. 424][45][46] For example, in murine models, Bacteroides thetaiotaomicron upregulates the transcription factor HIF-1α and antimicrobial peptide LL-37, initiating host innate immune response to inhibit C. albicans colonization. 44Notably, it is also said that B. thetaiotaomicron-processed mucins enhance C. albicans growth more than unprocessed mucins, and thus shaping its spatial distribution. 47Shortchain fatty acids (SCFAs), produced by bacterial metabolization and fermentation of non-digestible dietary fibers, typically Lactobacillus, are found to hinder hyphal morphogenesis and promote T cell response to C. albicans in mice, and finally inhibit biofilm formation and prevent invasion. 45,48acteria can exploit the microenvironment established by fungi for their growth.The hypoxic microenvironment generated by C. albicans in its biofilm is conducive to the growth of anaerobic bacteria such as Clostridium perfringens and Bacteroides fragilis . 40B. thetaiotaomicron is known to possess enzymes specialized in breaking down α-mannan, a typical cell wall component of yeasts. 49The growth of anaerobic Bacteroides species such as B. vulgatus and B. fragilis are also benefited from metabolizing fungal components in vitro. 50Several bacteria, including Streptococcal species, Fusobacterium nucleatum, and Helicobacter pylori, have been documented to exhibit synergistic relationships with C. albicans, amplifying their virulence. 51 study revealed that C. albicans influence the recolonization of cecal microbiota in mice treated with antibiotics.This resulted in an elevation of Enterococcus faecalis and a decrease in probiotic Lactobacillus strains. 52A subsequent investigation demonstrated that intestinal C. albicans in antibiotic-treated mice elicit altered expression of specific genes regarding inflammation and host response, resulting in a shift in bacterial diversity while not inducing gut inflammation, supporting the role of C. albicans in helping with the recovery of bacterial community. 53he dysbiosis of either of bacteria or fungi can cause the dysbiosis of the other.The administration of antibiotics can induce fungal dysbiosis and the overgrowth of C. albicans . 54Jiang et al. demonstrated that diverse fungal species can effectively substitute for enteric bacteria in addressing infectious and inflammatory disorders, including colitis resulting from the elimination of commensal bacteria. 55Van Tilburg Bernardes et al. illustrated that fungal colonization leads to significant alterations in the ecology of the bacterial microbiome.][58] In all, bacteria and fungi interact in various ways, forming polymicrobial biofilms and influencing each other's growth and virulence.Bacteria inhibit C. albicans through diverse mechanisms, while fungal biofilms can create favorable environments for anaerobic bacteria.Dysbiosis in one can disrupt the other, with fungi potentially substituting for bacteria in addressing inflammatory disorders.This interplay significantly alters microbiome ecology and can impact the course and severity of pathogenic status such as colonic inflammation.

Invasiveness and biofilm formation
Biofilms are closely packed communities of cells that attach to biotic and abiotic surfaces, including the intestine. 59,60C. albicans biofilms exhibit a highly organized structure, incorporating yeastforming cells, pseudohyphae, and hyphae enveloped by an extracellular matrix.These biofilms participate in the survival of C. albicans.C. albicans biofilms are typically formed following three stages: yeast cell adsorption and adherence, hyphae development with microcolony formation and extracellular matrix (ECM) production, and maturation with cell dispersal for potential dissemination to secondary infection sites. 61,624][65][66][67][68] The ECM is composed of self-produced extracellular biopolymers within the biofilm.Macromolecules, such as protein, lipid, and various polysaccharides including α-mannans and β-1,6-glucans, 69 as well as plentiful of host cells such as neutrophils exist, indicating host engagement in the formation and maintenance of these biofilms. 70uorum sensing factor farnesol, initially identified in high-density cultures, serves as a crucial inhibitor of hyphal formation and participates in the dispersal of biofilm cells of C. albicans . 71arnesol hinders the maturation of dendritic cells, and thus causing failure in inducing proper T cell response and innate immunity against C. albicans . 72Candida biofilms offer enhanced protection against the immune system and antifungal therapy compared to planktonic cells.Factors like hyphal morphology, ECM, farnesol, resistance-associated gene expression, fungal-bacteria interactions, persister cells, and biofilm extracellular vesicles all contribute to this mechanism. 61,73,74. albicans possess a strong propensity for invasion through the epithelial cells in their hyphae form, an important factor in their pathogenesis. 75,76Enzymes like enolase and phytase possess the ability to anchor to the membrane, contribute to hyphal growth, host fibronectin, and laminin binding, epithelial tissue damage, and nutrient acquisition. 77,78Although much have been concerned about Candida biofilm and infection, the relationship between fungal biofilm and carcinogenesis has seldomly been reported, except that in oral cancer. 79The specific features of biofilm, especially its resistance to host immune system, and strong capacity of invasion and dispersion may play a role in the tumorigenesis process.
Fungal species other than C. albicans are also capable of forming biofilms and causing biofilmrelated infection.Some Malassezia species such as Malassezia pachydermatis and Malassezia furfur are also found to be able to form biofilms in vitro, and these biofilms also possess the ability of pathogenesis and drug resistance. 80,81Aspergillus , 82 Cryptococcus, 83 and many other fungi also possess the ability to form biofilms and act differently on the pathological process.
In other words, biofilms are crucial for the survival of fungi, especially C. albicans, exhibiting structured layers composed of yeast, pseudohyphae, and hyphae within an extracellular matrix.
Factors including farnesol, ECM, and fungalbacteria interactions enhance C. albicans biofilm resistance against immunity and antifungal therapy.The invasiveness of the hyphae form of C. albicans contributes to its pathogenesis.While rarely reported, fungal biofilms may impact carcinogenesis, exemplified in oral cancer.

Candidalysin
Candidalysin is a cytolytic toxin produced by C. albicans that enables its capacity to damage and evade through host epithelium, and is encoded by the hypha-specific ECE1 gene. 84Candidalysin activates the MAPK pathway, triggering host immune responses such as heat shock protein (Hsp) activation, IL-6 release, and neutrophil recruitment. 85However, the detailed signaling caspase involved is still unknown.A recent study highlighted the significance of EGFR activation in candidalysin-induced MAPK signaling.This activation leads to the induction of the c-Fos transcription factor, ultimately triggering neutrophil recruitment through G-CSF and GM-CSF.The process relies on the release of EGFR ligands and calcium influx. 86While S. A. Nikou et al. mentioned another mechanism of candidalysin in oral epithelial cells that involves the activation of EGFR and c-Fos independent of MAPK. 87Rogiers et al. revealed the pivotal role of candidalysin in eliciting C. albicans to induce NLRP3 inflammasome responses, leading to the maturation and secretion of IL-1β in macrophages. 88Additionally, C. albicans mutants that are unable to transit from yeast to hyphae exhibited compromised TCRαβ + cell proliferation and diminished IL-17a expression in oral epithelial cells.This suggests a potential role of candidalysin in engaging in the recognition and induction of innate immunity of fungi as mentioned above that involves IL-1β and IL-17. 89

Acetaldehyde
C. albicans, as along with some Candia species other than albicans such as C. glabrata and C. tropicalis, are able to produce acetaldehyde (ACH) in vitro. 90CH is a genotoxic compound involved in ethanol metabolism, disrupting DNA repair processes and inducing oxidative stress.Dysbiosis can increase local ACH production by opportunistic pathogens, forming a causal link with oral and gastrointestinal carcinogenesis.High ethanol-derived ACHproducing Candida were more prevalent in oral cancer than non-oral cancer patients. 79

Indole
Indoles are alkalic molecules transformed by tryptophan, including indirubin, indolo (3,2-b) carbazole, and FICZ.They can be produced by Malassezia metabolism. 91,92Indole compounds are robust ligands and agonists of the aryl hydrogen receptor (AhR), and they are associated with skin carcinogenesis. 93Abundant AhR, malassezin, and indoles can be observed in M. furfur yeast strains causing skin infections, and is related to disrupted TLR-associated dendritic cell maturation. 92Kynurenine, a key oncometabolite in the tryptophan pathway linked to numerous diseases, serves as a ligand activating AhR in cells.Blocking kynurenine has proven to inhibit the growth of CRC cells in colon organoids. 94][97] Aspergillus species are able to produce aflatoxin that is a well-studied carcinogenic mycotoxin for hepatocellular carcinoma (HCC) development, and we will mention it in detail later.

Specific fungi in oncogenesis
Similar to bacteria, specific gut fungal genus, and species display an augmentation in the intestine of specific tumor patients, and they may contribute to tumor progression through different mechanisms (Figure 2).They are suspected to be a cause even if the tumor does not originate along the digestive tract, and the possibility of migration helps to explain this phenomenon.

Candida
Candida is commonly manifested as a commensal component of the human gastrointestinal tract.However, it can become pathogenic due to various host-related factors such as gut microbiome disruption, immunocompromised status, and significant metabolic changes.While candidemia is the most direct manifestation, Candida is also associated with multiple types of cancer.Its role in oral cancer is the most extensively explored, 98 with reported but inconclusive evidence of its involvement in other cancers including colorectal cancer, neck and cervical cancer.

Candida Albicans
C. albicans, in normal health status, presents as harmless native commensal microbiota colonizing mucosa at multiple sites, including mouth, skin, the gastrointestinal tract, vagina, and vascular system. 64,65,68However, it also acts as an opportunistic pathogen capable of causing severe and potentially fatal bloodstream infections, particularly in both immunocompromised and immunocompetent individuals.C. albicans can hematogenously disseminate, reaching multiple organs and potentially leading to serious complications.C. albicans displays significantly increased pseudohyphae morphogenesis, virulence factor secretion, and biofilm formation compared to certain non-albicans Candida species such as C. auris, C. tropicalis, C. parapsilosis, C. haemulonii, and C. glabrata, observed in vitro studies and in murine models. 99,100. albicans can potentially drive cancer progression through multiple mechanisms, such as the generation of carcinogenic byproducts, initiation of chronic inflammation, alteration of the immune microenvironment, activation of oncogenic signaling pathways, and interactions with bacteria.Here, we summarize the possible relationship between cancers and C. albicans in or derived from the gut.

Esophageal cancer. Esophageal cancer (EC) has been found to be associated with
Candida infection, of which C. albicans are the most relevant. 101It is more prevalent in patients with EC than in those with esophagitis (27% compared to 15%), Candida infection may be correlated to the development of EC. 102 On average, patients with fungal infections are 7 years younger than those with EC, suggesting fungi as potential triggers for EC initiation and development. 103ronic esophageal candidiasis is primarily observed in immunocompromised conditions, including chronic mucocutaneous candidiasis (CMC), which is a common clinical symptom of autoimmune polyendocrinopathy-candidiasisectodermal dystrophy (APECED).5][106] Patients with C. albicans may experience differing degrees of T-lymphocyte dysfunction, potentially increasing their susceptibility to esophageal cancer, proven by the fact that fungal depletion and administration, respectively, inhibits and accelerates ESCC development. 107Delsing and Koo suggested a relationship between STAT1 function deficiency, autoreactive CD4 + T cells, and chronic fungal infection.Koo et al. documented a family with CMC and a confirmed gain-of-function STAT1 mutation.The patients, who experienced CMC in childhood, presented severe oral and esophageal candidiasis and were at risk of developing oral cancer and/or ESCC. 106Delsing et al. expressed concern about nitrosamine production, and defective T-lymphocyte function against oncogenesis along with the STAT1 mutation in the development of esophageal carcinoma. 105Another potential mechanism could be the local production of ACH by C. albicans, given the significant microbial composition similarity between the esophageal mucosa and the oral cavity. 108,109

Gastric cancer.
H. pylori has long been acknowledged as the most important risk factor for gastric cancer (GC).However, whether other microbials take their part in the development of GC remains largely unexplored.
In 2014, Sano et al. showed that infection caused by orally or intravenously administrated C. albicans leads to chronic hyperplastic candidiasis and ultimately induces oncogenic processes in the stomach in diabetic rat models. 110Zhong et al. later substantiated the hypothesis by revealing the notable fungal dysbiosis in the intratumor biopsy samples, characterized by a significant increase of C. albicans abundance and a minor increase in other species such as Fusicolla acetilerea and Arcopilus aureus . 111This enrichment of C. albicans is also shown in a pan-cancer analysis. 112Furthermore, a case report mentions the possibility that genetic mutations and C. albicans infection corporately induce GC development, both leading to defective production of IL-17. 113Intravacuolar H. pylori, found within Candida cytosols in mice, indicates endosymbiosis function of C. albicans.This protective environment within C. albicans vacuoles may participate in promoting the virulence and survival of H. pylori, potentially contributing to tumor development. 114ontrarily, Zhang et al.'s in silico study found no difference in Candida species abundance between tumor lesions and surrounding normal tissues in 61 GC patients.Instead, a significant enrichment in the Solicoccozyma was observed. 115Furthermore, a prospective cohort study conducted by Ndegwa et al. revealed no associated risk of Candida-related oral lesions with the development of GC. 116

Colorectal cancer.
Until now, there have been no consistent perspectives on the role of C. albicans in CRC development.Patients with inflammatory bowel disease are always regarded as a high-risk population of CRC.8][119] There are studies showing no increase in albicans abundance neither in CRC nor in adenoma patients in their intratumor biopsy samples. 120,121Gao et al. even reported the considerable decrease in the number of Candida species in CRC. 120Furthermore, Jin et al.'s research revealed that, despite the fact that high-fat diet is associated with dysbiosis and alteration of multiple bacterial and fungal species, the abundance of gut Candida was reduced other than increased in mice that developed CRC. 122nti-fungi immunity, especially for C. albicans, sensed via Dectin-Syk-CARD9 pathway, is found to be a protective factor for colitisassociated cancer (CAC).In Card9 −/− mice, fungal dysbiosis was observed, leading to increased STAT3 activation-an essential transcription factor for MDSCs and a significant driver for CRC tumorigenesis. 26,123In Zhu's study, the elevated C. albicans load in Dectin-3 −/− mice load triggers glycolysis in macrophages and IL-7 secretion.The IL-22 production followed by IL-7 production in innate lymphoid cells are implemented through the AHR and STAT3 experiments. 124oss of Dectin-3 also leads to decreased inflammasomes and IL-18 activation, leading to impaired CD8 + T cell function, decreasing epithelial barrier restitution and IFN-γ production in mice. 125lthough distinct relationship has not been discovered, bacterial-fungal interactions can be highly suspected in the tumorigenesis process.Sovran et al. proposed a bacterial-fungal interaction affecting DSS-induced colitis in mice.Antibiotic treatment altered susceptibility and the impact of fungi on colitis, with C. albicans exacerbating colitis in untreated settings. 57

Liver cancer.
Recently, the possible link between liver disease and C. albicans has been taken into consideration, and mechanisms explored include translocation of β-glucan, 126 Th17 cells, 127 and candidalysin 128 derived from or secreted by intestinal albicans, as well as the activation of Dectin-1 in Kupffer cells, and subsequent increase in IL-1β expression. 129Nevertheless, still little is known about whether gut fungi count in HCC, although some bacterial species are found to be correlated. 130he gut mycobiome showed decreased diversity in HCC patients among various studies. 131,132espite this, C. albicans has undergone an enrichment in the gut of HCC patients.In tumor-bearing mice, C. albicans was observed to promote HCC by reprogramming NLRP6-dependent cancer cell metabolism. 131C. albicans was found to be significantly more abundant in late stage HCC. 132

Usami et al. suggested a likelihood between intestinal
Candida and serum fatty acid metabolism in HCC.They found strong positive correlations (coefficients 0.81 and 0.88) between serum EPA (eicosapentaenoic acid), EPA/AA (arachidonic acid) ratio, and fecal Candida in the liver cirrhosis (LC) group of 46 HCC patients. 133s a commensal microbe, C. albicans exhibits higher virulence compared to other Candida species, contributing to cancer progression via carcinogenic byproducts, chronic inflammation, and altered immune microenvironment.In esophageal cancer, C. albicans infection correlates with younger onset and autoimmune conditions; gastric cancer may be influenced by fungal dysbiosis and the protective effect of C. albicans on H. pylori, potentially promoting oncogenesis; in colorectal cancer, the role of C. albicans are most studied with the involvement of Dectin-Syk-CARD9 pathway, with bacterial-fungal interactions suspected; liver cancer studies suggest C. albicans enrichment in HCC patients, impacting metabolism and disease progression.Also, C. albicans colonized in the mucosa other than the gut can influence site-specific tumors with various mechanisms.C. albicans in the oral cavity is related to oral pre-cancer lesions and can directly induce oral cancer through IL-17 activation, biofilm formation, oncogenic metabolites, and mycotoxin production, such as candidalysin, nitrosamine, and ACH. 76,134Interestingly, oral albicans have been found to be correlated with blood malignancy in several cohorts. 135,136

C. tropicalis
8][139] Until now, the majority of the observed mechanical processes are associated with Dectin-Syk-CARD9 pathway.C. tropicalis is specifically increased in Dectin-3 −/− and Card9 −/− CAC mice, and antifungal treatment ameliorated CAC in the previous study. 26,125Deficiency of Dectin-3 impairs phagocytic and fungicidal abilities of macrophages in C. tropicalis and thus promotes colitis. 125The increase of C. tropicalis is demonstrated to enhance the accumulation of MDSCs in Card9 −/ − mice. 26However, further research indicated that C. tropicalis promotes tumorigenesis through influencing aerobic glycolysis by the Dectin-3-Syk-PKM2-HIF-1α glycolysis signaling axis, which involves MDSCs. 138Toxic products such as NADPH oxidase 2 and reactive oxygen species (ROS) are produced and lead to T cell dysregulation. 138Their further investigation revealed that the C. tropicalis facilitates the NLRP3 inflammasome production through the Dectin-3-glycolysis pathway, and finally leads to the increase in IL-1β production on MDSCs.The wholistic process involves activation of several transcription factors and signaling molecules, including STAT1, pro-caspase-1, mitochondrial ROS, and finally inducing IL-1β production.The elevation of these participants to immune pathways may suggest another target for CRC treatment. 17

Malassezia
To our recent knowledge, Malassezia is composed of 18 species, most of which colonize the skin, but can also colonize other mucosal surfaces such as human gut, as well as abiotic surface that form biofilms. 141 Malassezia genus has been reported to be the second most abundant genus among all human stools in proportions from 2 to 4%, while M. restricta (more than 80%) and M. globosa (36%) account for the most prevalent species. 142,143

CRC and liver cancer
While Malassezia has been extensively studied as an important contributor to the tumorigenesis of skin and breast cancer due to its skin colonization, 144 there is relatively limited research on its impact on cancers resulting from gut colonization.
An increased abundance of Malassezia spp.and fungal dysbiosis was found in IBD patients, 117 as well as in CRC patients. 145,146Interestingly, one study also mentioned an increased abundance of Malassezia species in HCC patients compared with cirrhosis and healthy controls. 132nfortunately, few studies have been dedicated to the underlying mechanisms of gut Malassezia in gastrointestinal oncogenesis so far, and mountainous research needs to be done to clarify the exact relationship.A possible mechanism may lie in the metabolism of tryptophan into indole that is associated with kynurenine production. 94

Pancreatic cancer
While there is extensive discussion on the role of bacteria, such as Fusobacterium sp., Enterobacteriaceae, and H. pylori, in pancreatic cancer tumorigenesis, limited efforts have been paid on the involvement of fungi in this oncogenic process. 147Research on tumorigenesis in KC mice (p48 cre ;LSL-Kras G12D ), a model expressing oncogenic Kras in pancreatic progenitor cells for slow progressive pancreatic ductal adenocarcinoma (PDA), demonstrated significant fungal dysbiosis in the pancreas, with Malassezia spp.being the predominant genus. 148Unlike the gut or normal pancreas, both mouse and human PDA mycobiomes featured increased Malassezia species.Notably, Malassezia in the pancreas tissue is shown to have migrated from the gut. 148ectin-1 is not normally expressed in human pancreatic cells. 149However, it has been identified in association with inflammation caused by Malassezia folliculitis . 14Dectin-1 functions, particularly in fungal infections, by initiating the Syk-CARD9-NFκB pathway and activating the downstream caspase-8-ASC complex, ultimately leading to the production of IL-1β. 12,150,151Alam et al. discovered that this Dectin-1 pathway contributes to the pathogenic pathway of Malassezia through activation of cytokines IL-33 from PDA cells, a downstream target of Kras G12D in tumorbearing mice.Synergistically, biochemical factors like ROS can stimulate and amplify IL-33 secretion in collaboration with the mycobiome. 149his IL-33 secretion may participate in triggering a type 2 immune response, hastening the pancreatic cancer progression along with reducing survival. 152ykut et al. proposed that Kras oncogenic murine models exhibit fungal dysbiosis, exacerbating tumor progression through the activation of the C3 complement cascade via recognition and ligation with mannose on cell walls of Malassezia through the mannose-binding lectin.The complement system then participates in the corruption of body immunity, expansion, and apoptosis of tumor cells and therapeutic resistance. 147,148n a comparative cohort study examining 16 pancreatic cancer patients who achieved longterm survival (at least 4 years) postpancreatectomy without recurrence, with eight patients after pancreatectomy and chemical therapy as the control group, we found no significant difference in Malassezia abundance in their fecal samples, indicating that Malassezia may not influence the prognosis. 153ncreased Malassezia spp.abundance has been observed in IBD, CRC, and HCC patients, suggesting its potential involvement in tumorigenesis.Mechanisms may involve tryptophan metabolism and IL-33-mediated inflammation through the complement cascade activation.However, the clinical importance of Malassezia has not been proven yet, awaiting further studies.

Colorectal cancer
An in silico multi-cohort analysis revealed that A. rambellii and other five fungal species were enriched in CRC patients compared with patients with adenoma or normal controls.The enrichment of A. rambellii was validated both in vivo and in vitro, with another interesting finding that interactions occur among the enriched microbes.The enriched fungi showed significant co-current features, and the A. rambellii also exhibited a correlation with Fusobacterium nucleatum and P. micra (z score −5.95 and −5.07 respectively). 154. rambellii administration promoted tumor growth, weight, and proliferation both in vitro and in a xenograft mice model.They further announced that the panel combing both fungi and bacteria better distinguishes CRC patients from health controls and adenoma patients than pure fungal or bacterial panels. 154A. rambellii and other two species were among the top 3 fungal species that were significantly enriched in CRC according to Gao et al.Other Aspergillus species, that is A. ochraceoroseus and A. flavus, were also found to have increased in abundance in CRC. 120

Liver cancer
Aflatoxins have been one of the earliest discovered and studied mycotoxins worldwide, with countless studies reporting their potential pathogenic and carcinogenic effects majorly focusing on two species: A. flavus and A. parasiticus . 132The majority of aflatoxins contributing to liver cirrhosis and HCC are of exogenous origin, particularly in developing countries.Aflatoxins are most commonly absorbed through ingestion and rapidly cleared from blood.AFB1 has been experimentally shown to possess the capability to damage intestinal epithelial cells, leading to the disruption of intestinal structure. 155This leads to an influx of immune cells, which may exacerbate tissue damage and impair the absorption of nutrients, particularly vitamins A and E. 156 Upon absorption, AFB1 is mainly distributed in the liver, while lesser amounts are found disseminating into kidneys and mesenteric vein.Metabolized by the CYP450 system, AFB1 forms DNA adducts, which is its major mechanism for tumorigenesis.AFB1 also induces ROS and inhibits protein, RNA, and DNA synthesis, ultimately leading to apoptosis in microglia via NF-kB activation in oxidative stress. 96. rambellii, A. flavus and A. parasiticus exhibit enrichment in tumors, indicating the potential role of Aspergillus in tumor development.AFB1 produced by A. flavus and A. parasiticus significantly contributes to HCC development by damaging intestinal epithelial cells, impairing nutrient absorption, and inducing oxidative stress, leading to apoptosis and tumorigenesis.

Other fungi
Some fungi species other than those mentioned above are reported to show differences in abundance in cancer patients, despite the fact that they only take up a little account of the gut fungal composition.Chang et al. mentioned that apart from C. albicans, Torulopsis glabrata, Torulopsis tomata, C. tropicalis, C. krusei, and C. parepsilosishave have also been isolated from esophageal samples. 103Considering GI tumors with intratumor lesions, C. albicans, C. tropicalis, C. dubliniensis, and other Candida species could all be distinguished with an increase in abundance, with interactions with certain bacterial and fungal species, such as Lactobacillus gasseri and S. cerevisiae . 112ultiple fungi species, although not directly implicated in tumor progression, are potentially associated with exacerbating diseases that have a high likelihood of progressing into cancer.
Phoma, another opportunistic pathogen, is found to cause lung mass 36 and subcutaneous mycosis. 121. metapsilosis M2006B demonstrated the ability to alleviate experimental colitis in a large-scale human intestinal fungal cultivation and functional analysis. 117

Fungi as biomarkers
Several studies have demonstrated the possibility of utilizing fungi as biomarkers for tumor screening and diagnosis due to the significant difference in fungal composition compared with the healthy control. 146,157n important parameter is the Basidiomycota: Ascomycota ratio (B/A ratio), a significant increase in B/A ratio is found concerning CRC and ulcerative colitis, 146,158 although this can be controversial among different studies. 145Models incorporating diverse microbiomes have been proposed for predicting diseases like colon adenoma and CRC.They exhibit varying compositions, resulting in differences in accuracy that can be estimated by the area under the receiver-operating characteristic curve (AUROC).However, there are studies showing no difference in fungal composition in CAC. 159Decreased diversity of fungi indicating fungal dysbiosis is found in polyp patients, supporting the role of fungi in chronic tumorigenesis process. 121,145Luan observed a correlation between malignancy potential of adenoma, and disease stage with changes in fungal composition, highlighting specific fungal species composition at each stage. 121Gao et al. reported a non-significant increase in M. restricta, with Leucoagaricus_sp_SymCcos and fungal_sp_ARF18 undergoing depletion in adenomas. 120In the study conducted by Cocker et al., 14 fungal biomarkers achieved an AUROC of 0.93 that effectively distinguished CRC from controls.However, these fungal markers only achieved AUROCs of 0.63 in classifying adenoma subjects from controls. 146Another study discovered that 16 multi-domain markers including 11 bacterial, four fungal, and one archaeal feature achieved good performance in diagnosing patients with early-stage CRC with an AUROC of 0.78. 157ao et al. regarded Taxomyces andreanae, A. rambellii, Lachancea_waltii, fungal_sp_ARF18, and Phanerochaete chrysosporium as the top five most important biomarkers with the AUROC of 0.926. 154ome other cancers were also evaluated for the potential of utilizing gut fungi as tumor biomarkers, including GC and HCC. 111,115,132umerous studies have agreed on the potential that fungal-bacterial interplay influences the tumor development.Cocker et al. announced a significant increase in amount and detrimentality, but also more antagonistic bacterial-fungal interplay in healthy patients compared with CRC patients (p < .00001),very similar with the discovery by Liu et al., as well as our previous research, demonstrates that an addition of bacterial biomarkers to the fungal marker can further increase the AUROC of our random forest model in predicting the effect of immune checkpoint inhibitors. 146,157,160wo large pan-cancer studies manifested a significant relationship between intra-tumor fungal ecologies and the tumor type and the fungalfungal interaction or fungal-bacterial interaction as potential indicator of prognosis, such as the increased Candida-to-Saccharomyces ratio in latestage metastatic CRC, and specific fungal-bacterial-immune clusters. 112,161

Chemotherapy
Researchers have discovered the effect of gut microbiota, especially gram-positive bacteria, in reducing the pathogenic Th17 cell response and rescuing cyclophosphamide resistance. 162,163New strategies have been discovered targeting gut microbiota to enhance the efficacy of chemotherapy, including bacterial derivatives and phageguided nanotechnology. 164,165ew studies have been dedicated to the association of the fungal community with the efficiency and resistance of tumor chemotherapy.C. tropicalis is associated with CRC and is shown to promote chemoresistance via lactate production and MLH1 inhibition.Inhibiting lactate mitigates the resistance via the GPR81-cAMP-PKA-CREB pathway, regulating MMR protein expression. 139

Fecal microbiota transplantation
Fecal microbiota transplantation (FMT) is a transformative method that effectively alters recipients' gut microbiota by transferring a diverse consortium of host-adapted microbial species, encompassing bacteria, bacteriophages, fungi, and their metabolites. 166][169][170] FMT is able to reverse the reduction in fungi taxa and increase in C. albicans in recipients with CDI and ulcerative colitis. 158,167Overloading C. albicans in donor stool correlates with reduced FMT efficacy in CDI patients. 167Meanwhile, recipients with relatively high abundance of gut C. albicans respond better to FMT than controls in the condition of ulcerative colitis, and the mechanism may involve the beneficial condition for bacterial diversity recovery. 158It is also found to increase the abundance of certain fungi such as Aspergillus in CDI 167 and Schizosaccharomyces pombe in ulcerative colitis 171 after FMT.
The application of FMT has not extended to the treatment of malignancies until now, except for some attempts at improving the efficacy of immunotherapies.Wang et al. presented two case series of immune checkpoint inhibitor-associated colitis successfully treated with FMT, restoring gut microbiota balance, substantially decreasing cytotoxic T cells, accompanying the concomitant increase of regulatory T cells. 172Yet, since only two patients were treated and no control groups were involved in the study, more valid evidence should be given to prove its efficiency and safety. 172he long-time safety of FMT has not been proven, although studied for years.The known possible adverse events are only limited to mild side effects such as abdominal cramping, bloating, and sore throat, with one death discovered attributed to FMT. [173][174][175] More clinical trials and cohorts should be done to explore all the possible adverse effects to ensure its safety.

Fungal-derived products
Bacterial-associated products, including prebiotics, probiotics, and synbiotics, are the most commonly acknowledged microbial adjustment products that may work in tumor prevention and treatment. 176Meanwhile, fungal-derived products are also worth mentioning.Citrinin is a mycotoxin derived from polyketides, and it is produced by various fungal strains, including those belonging to the genera Monascus, Aspergillus, and Penicillium.It has been studied for a long time for its potential antineoplastic effect by inducing cell death, reducing DNA repair capacity, and inducing cytotoxic and genotoxic effects. 177,178β-glucan administration, whether oral or systemic, has shown promising antineoplastic effects in multiple malignancies including breast cancer, colon cancer, and melanoma, exhibiting benefits in metastasis treatment.][181] Marine-derived Aspergillus is capable of producing metabolites that are potentially tumoricidal.Asperphenin A inhibits CRC growth by triggering microtubule disassembly and cell cycle arrest, and also has anti-proliferative effect in multiple tumors. 182Preussin displayed cytotoxic and antiproliferative activities in breast cancer cell lines. 183β-Galactosidases, known as one of a kind of prebiotics, function as lysosomal exoglycosidases involved in glycoconjugate metabolism.This enzyme can also be isolated from Aspergillus Terreus to effectively promote the proliferation of MCF-7 breast cancer cells in vitro. 184

Radiotherapy
Whether microbiome affects radiotherapy has not been clearly comprehended until now.Commensal microbiomes may differently influence the response to radiotherapy.Depletion of fungi is beneficial for radiotherapy whereas antibiotic-mediated bacterial depletion reduces responsiveness.The immune responses included in these effects are orchestrated by Dectin-1. 185

Immunotherapy
Immunotherapy is revolutionizing tumor treatment.Strategies have been proposed to augment the potency of drugs and immune generation.Response to PD-1 blockade treatment was observed to have a positive correlation with increasing abundance of Akkermansia after FMT in murine models, followed by increased recruitment of CCR9 + CXCR3 + CD4 + T cells. 186FMT also shows positive results in counteracting tumor microenvironment and rescuing anti-PD-1 sensitivity in clinical trials of melanoma. 187Β-glucan and its nanoparticles are demonstrated to manipulate the tumor microenvironment, thereby augmenting the response to immunotherapy. 188Our recent study also demonstrated the gut bacterialfungi interplay and the possibility of fungi as biomarker for efficacy of immunotherapy. 160verall, the usage of fungi in the tumor treatment is almost a brand-new field.Recent studies majorly focus on the potential causal relationships of the fungi with the effectiveness, efficacy, and resistance of tumor treatment.Some specific fungi species and fungal products are mentioned, including C. tropicalis that promotes chemoresistance in CRC, some fungal-derived products like citrinin and β-glucan exhibiting antineoplastic effects, and Aspergillus metabolites that show promise in CRC treatment.But much more about fungi and cancer treatment awaits to be found, especially in the field of FMT and immunotherapy, which are the revolutionizing medical techniques and therapies for disease treatment.

Conclusion
Although fungi and cancer have been widely studied separately for years, the cancer mycobiome field remains at its starting-line.The techniques and database are far from united and integrated, and there are large amounts of unexplored gut fungi beyond our knowledge.The sample type and size vary greatly between studies and significant amounts of unclassified taxa were mentioned.These limitations have greatly disturbed scientists to find the actual causal relationship between cancer development and fungal changes.Nonetheless, recent studies still help with the discovery of the carcinogenic power of pathogenic fungi, functioning through immune activation, metabolites, toxin production, bacterialfungal interaction, or biofilm formation.Although the validity, feasibility, and security of the present strategies and investigation methods involving gut mycobiome need to be further addressed, the application of gut fungi to the screening, prevention, and treatment of cancers is a promising field well-worth dedication.

Figure 2 .
Figure 2. Fungi and their roles in specific tumors.Shown are the various mechanisms through which major gut fungi species are able to influence the specific cancers, including the immune responses, cytotoxins and metabolites, and bacterial-fungal interactions A new potential mechanism is asserted by Qu et al. that C. tropicalis can interfere with tumor PD-1 expression by increasing tumor cell autophagy and thus promote CRC progression.140